Original Article

Stage-Specific Profiling of Transforming Growth Factor-β, Fibroblast Growth Factor and Wingless-int Signaling Pathways during Early Embryo Development in The Goat Pouria HosseinNia, Ph.D. 1, 2, Mojtaba Tahmoorespur, Ph.D.1, Sayyed Morteza Hosseini, D.V.M.2, Mehdi Hajian, M.Sc.2, Somayeh Ostadhosseini, D.V.M.2, Mohammad Reza Nasiri, Ph.D.1, Mohammad Hossein Nasr-Esfahani, Ph.D.2* 1. Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran 2. Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

*Corresponding Address: P.O.Box: 8159358686, Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran Email: [email protected] Received: 16/Oct/2014, Accepted: 23/Nov/2014

Abstract

Objective: This research intends to unravel the temporal expression profiles of genes involved in three developmentally important signaling pathways [transforming growth factor-β (TGF-β), fibroblast growth factor (FGF) and wingless/int (WNT)] during pre- and peri-implantation goat embryo development. Materials and Methods: In this experimental study, we examined the transcripts that encoded the ligand, receptor, intracellular signal transducer and modifier, and the downstream effector, for each signaling pathway. In vitro mature MII oocytes and embryos at three distinctive stages [8-16 cell stage, day-7 (D7) blastocysts and day-14 (D14) blastocysts] were separately prepared in triplicate for comparative real-time reverse transcriptase polymerase chain reaction (RT-PCR) using the selected gene sets. Results: Most components of the three signaling pathways were present at more or less stable levels throughout the assessed oocyte and embryo developmental stages. The transcripts for TGF-β, FGF and WNT signaling pathways were all induced in unfertilized MII-oocytes. However, developing embryos showed gradual patterns of decrease in the activities of TGF-β, FGF and WNT components with renewal thereafter. Conclusion: The results suggested that TGF-β, FGF and WNT are maternally active signaling pathways required during earlier, rather than later, stages of pre- and periimplantation goat embryo development. Keywords: Goat, Gene Expression, TGF-β, FGF, WNT Cell Journal(Yakhteh), Vol 17, No 4, Jan-Mar (Winter) 2016, Pages: 648-658

Citation:

HosseinNia P, Tahmoorespur M, Hosseini SM, Hajian M, Ostadhosseini S, Nasiri MR, Nasr-Esfahani MH. Stage-specific profiling of transforming growth factor-β, fibroblast growth factor and wingless-int signaling pathways during early embryo development in the goat. Cell J. 2016; 17(4): 648-658.

Introduction Pre-implantation embryo development is a characteristic feature of mammalian embryo development which encompasses a series of crucial events suchas the transition from oocyte to embryo, first cell divisions, and establishment of cellular contacts. These processes are under strict control of spatial and temporal regulation of gene expression,

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cell polarization, and cell-cell interactions (1). The transcriptional circuitry that regulates embryo development comprises several hundred genes responsible for cell division, growth, differentiation, polarity, and apoptosis of embryonic cells. By combining several functions, such as cross-linking and other interactions, these genes

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provide pathways to form a complicated network of interactions that take shape in the context of various cell-signaling pathways which include fibroblast growth factors (FGF), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PtdIns3K)⁄protein kinase B (PKB), also known as Akt and Janus-activated kinase (JAK)⁄signal transducer and activator of transcription (STAT), the wingless/int (WNT)⁄b-catenin pathway, notch, bone morphogenetic protein (BMP)-Smad, and hedgehog (1). Pluripotency and self-renewal in the absence of differentiation are three fundamental traits of embryonic stem cells (ESCs) which are mostly maintained by the core transcription triad-OCT4, SOX2, and NANOG (2). Importantly, it has been established that the pluripotency transcription triad is highly responsive to upstream and downstream signals induced by WNT, transforming growth factor-β (TGF-β) and FGF signaling pathways. A functional WNT signaling system operates in the pre-implantation embryo and activation of the canonical pathway affects embryonic development in bovines (3), ESC self-renewal in humans and mice (4), as well as tumor progression (5). It is well established that phosphorylation inhibition of TGF-β signaling by SB(4-(5-Benzol[1,3] dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)benzamide hydrate, 4-[4-(1,3-Benzodioxol-5-yl)5-(2-pyridyridyl)-1H-imidazol-2-yl]-benzamide hydrate) supports mouse ESC self-renewal in the differentiation (6). The growth of primed stem cells are dependent on the FGF signaling pathway and notably, dual inhibition of extracellular signalregulated protein kinases 1 and 2 (ERK1/2) and glycogen synthase kinase 3 (GSK3), designated as 2i, has been shown to improve the efficacy of ESC-derivation in mice (7). Despite two decades of effort, derivation of authentic ungulate ESCs remains challenging for embryologists. To date, ESCs have been successfully isolated only in rodents and primates. A clear understanding of the signaling pathways that regulate early embryo development will greatly benefit the current understanding of developmental biology and approaches to capture pluripotent stem cells in vitro. The current study has attempted to investigate the dynamics of expression of components from the three developmental signaling pathways (WNT, TGF-β,

and FGF) at four distinctive stages of goat embryo development: i. Unfertilized in vitro mature (MII)-oocytes, ii. 8-16 cell stage which coincides with the stage concomitant with zygote genome activation in the goat, iii. Day-7 (D7) blastocysts and IV. Day-14 (D14) blastocysts.

Materials and Methods Chemicals and media Unless otherwise stated, all chemicals were obtained from Sigma Chemical Co. (USA) and media from Gibco (Grand Island, USA). Selection of gene sets Due to the lack of sufficient data in the goat species, we searched related studies in humans (8), mice (9), bovine (10) and porcine (11) for the conserved upstream and downstream components of the TGF-β, FGF, and WNT signaling pathways. Accordingly, we selected 4 transcripts for the TGF-β (Bmpr1a, Alk4, Sdma1 and 5, Id3), 4 transcripts for the FGF (Lifr1, Akt, Fgf4, Erk1, Cdc25a) and 3 transcripts for the WNT (Fzd, Ctnnb, c-Myc) signaling pathways. The genes involved in both the core pluripotency triad (Oct4, Nanog, Sox2) and cell lineage commitment (Rex1, Cdx2, Gata4) were also considered. We took into consideration the lack of a previous report or database on gene sequences of many of these genes and designed the primers according to the conserved regions of these markers in bovine, ovine, humans and mice. For Erk1, Alk4, Bmpr1, Fgfr4 and Lifr1, portions of the cDNA were initially sequenced and registered in the NCBI site under the following accession numbers: KC687077 (http://www.ncbi.nlm.nih.gov/nuccore/ KC687077), KF039752 (http://www.ncbi.nlm.nih.gov/nuccore/ KF039752), KF039753 (http://www.ncbi.nlm.nih.gov/nuccore/ KF039753), KF039754 (http://www.ncbi.nlm.nih.gov/nuccore/ KF039754), and KF356183 (http://www.ncbi.nlm.nih.gov/nuccore/ KF356183). Specific primers were subsequently designed from these recognized sequences (Table 1). CELL JOURNAL(Yakhteh), Vol 17, No 4, Jan-Mar (Winter) 2016

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Table 1: Specific real-time primers designed for gene sequences

Gene

Primer sequences

Length of PCR product

TM

Lifr1

F: ATTTTTCGGTGTATGGGTGC

117

56

116

58

152

62

140

60

136

60

198

58

160

54

98

60

96

56

94

61

89

59

160

61

182

54

R: CAGATGTATCCTCAACGGTA Bmpr1

F: CCTGTTCGTCGTGTCTCAT R: CTGGTGCTAAGGTTACTCC

Alk4

F: TCTCCAAGGACAAGACGCTC R: ACGCCACACTTCTCCAAACC

Smad1

F: TCACCATTCCTCGCTCCCT R: AAACTCGCAGCATTCCAACG

Smad5

F: ACAGCACAGCCTTCTGGTTC R: GGGGTAGGGACTATTTGGAG

Id3

F: CGGCTGAGGGAACTGGTA R: CCTTTGGTCGTTGGAGATG

Ctnnb

F: AGTGGGTGGCATAGAGG R: CACAGGTAGCCCGTAG

Akt

F: TTCAGCAGCATCGTGTGGCA R: TCATCAAAATACCTGGTGTCCG

Oct4

F: GCCAGAAGGGCAAACGAT R: GAGGAAAGGATACGGGTC

Rex1

F: GCAGCGAGCCCTACACAC R: ACAACAGCGTCATCGTCCG

Fzd

F: CATCGGCACTTCCTTTATCC R: GCTTGTCCGTGTTCTCCC

C-myc

F: CAACACCCGAGCGACACC R: GCCCGTATTTCCACTATCCG

Sox2

F: ATGGGCTCGGTGGTGA R: CTCTGGTAGTGCTGGGA

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Table 1: Continued

Gene

Primer sequences

Length of PCR product

TM

Fgfr4

F: GCTGACTGGTAGGAAAGG

193

56

137

54

204

58

128

64

144

53

119

58

146

60

R:AGTGGCTGAAGCACATCG Nanog

F: GATTCTTCCACAAGCCCT R:TCATTGAGCACACACAGC

Erk1

F:TCAAGCCGTCCAACATCCT R:CGACCGCCATCTCAACC

Gata4

F:TCCCCTTCGGGCTCAGTGC R:GTTGCCAGGTAGCGAGTTTGC

Cdx2

F:CCCCAAGTGAAAACCAG R:TGAGAGCCCCAGTGTG

Cdc25a

F:TGGCAAGCGTGTTATCGTG R:GGTAGTGGAGTTTGGGGTA

ACTB

F:CCATCGGCAATGAGCGGT R:CGTGTTGGCGTAGAGGTC

PCR; Polymerase chain reaction and TM; Melting temperature.

In vitro production of goat embryos This experimental study was conducted from 2011-2014.We used 850 goat ovaries that hadbeen derived from local breed does (Isfahani, Najdi) immediately after slaughter. The procedure for in vitro production of goat embryos has been previously described (12). In brief, goat ovaries were used for in vitro maturation of cumulus-oocyte complexes (COCs) in tissue culture medium-199 (TCM199) plus 10% fetal calf serum) FCS, (2.5 mM sodium pyruvate, 100 IU/mL penicillin, 100 µg/mL streptomycin, 10 µg/mL follicle stimulating hormone (FSH), 10 µg/mL luteinizing hormone (LH), 1 µg/mL estradiol-17β, and 0.1 mM cysteamine under mineral oil for 20-22 hours at 39˚C, 5% CO2,

and maximum humidity. Next, they were divided into six groups and placed in 20 µl droplets that consisted of a modified formulation of synthetic oviductal fluid (mSOF) (12) and maintained at 39˚C, 6% CO2, 5% O2, and maximum humidity for embryo development. The MII oocytes at 20-22 hours post-maturation, day 3 (D3) developing embryos at the 8-16 cell stage, and day 7 (D7) blastocysts were collected, washed three times in phosphate-buffered saline (PBS), collected in pools of 60 (oocytes), 35-40 (D3 developing embryos), and 20 (D7 blastocysts) in 500 µL microtubes that contained lysis buffer RLT. They were subsequently frozen and stored at -70˚C until RNA extraction. All oocyte and embryo pools used for RNA extractions were CELL JOURNAL(Yakhteh), Vol 17, No 4, Jan-Mar (Winter) 2016

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collected and analyzed in triplicate. This system of embryo development had adequate rates of in vitro embryo development with cleavage rates between 85 to 92% and blastocyst rates between 40-45%.

oped spherical D14 embryos were pooled for RNA extraction as previously described.

Derivation of day-14 embryos

The procedure for quantitative real-time PCR (qRT-PCR) has been previously described (14). In brief, total RNA from MII-oocytes, 8-16 (D3), blastocysts (D7) and elongating embryos (D14) was extracted with the RNeasy Micro kit (Qiagen, Canada) followed by treatment with DNase I (Ambion, Canada) according to the manufacturer’s protocol. RNA quality and quantity were determined using a WPA Biowave spectrophotometer (Cambridge, United Kingdom). For reverse transcription, 10 µl of total RNA was used in a final volume of a 20 μl reaction that contained 1 µl of random hexamer, 4 μl RT buffer (10x), 2 µl of dNTP, 1μl of RNase inhibitor (20 IU), and 1μl of reverse transcriptase (Fermentas, Canada). Reverse transcription was carried out at 25˚C for 10 minutes, 42˚C for 1 hour and 70˚C for 10 minutes.

In order to extend the in vitro culture of goat blastocysts, we prepared a feeder later of caprine fetal fibroblasts (CFF) as described by Behboodi et al. (13). For this purpose, the CFF line was derived from three 40-day male fetuses surgically from donors. A single-cell suspension was prepared by mincing fetal tissue and culturing the tissue in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% FBS, 0.25% amphotericin-B, 1% penicillin-streptomycin, and 1% gentamicin in 25 cm2 culture flasks at 37˚C and 6% CO2 until the appearance of a confluent monolayer from D4 onwards. The monolayer was trypsinized and further cultured for proliferation of the CFF source. Each passage took approximately 3-4 days until confluency. Passages 2-4 CFFs were treated with mitomycin (10 mg/mL) for 2 hours. Mitomycin treated cells were washed twice with DMEM and treated with trypsin-Ethylenediaminetetraacetic acid (EDTA) 0.25% (Supplemented by EDTA) and gently pipetted the confluent monolayer in order to obtain single cells. Cells were then seeded at 1×105 cells/ml in 100 µl DMEM drops in the vicinity of a feeder-free 100 µl droplet of DMEM supplemented with 10% FBS, 1% Lglutamine, 1% non-essential amino acids, and 0.1% β-mercaptoethanol under mineral oil. We transferred 5-6 D7 blastocysts to each 100 µl droplet of feeder-free DMEM. By the aid of the tip of a draw pipette glass, the DMEM drops that contained blastocysts were gently connected to their adjacent DMEM that had a CFF monolayer. This joined culture system provided the beneficial effects of a feeder layer for extended in vitro embryo culture, but prevented attachment and flattening of the elongating blastocysts. The joined droplets were refreshed every other day until D14 of embryo development when pools of 7-10 well-develCELL JOURNAL(Yakhteh), Vol 17, No 4, Jan-Mar (Winter) 2016

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RNA extraction and real time-polymerase chain reaction

Quantitative analysis of transcripts by real time-polymerase chain reaction

The transcripts abundance of the mentioned genes (Table 2) and ACTB as the housekeeping gene were analyzed with real-time RTPCR. Briefly, total RNA from the oocytes, D3 embryos, D7 blastocysts, and D14 blastocysts were extracted. Each of the RNA samples was used for cDNA synthesis. Real-time RT-PCR was carried out using 1 µl of cDNA (50 ng), 5 μl of the SYBR Green/0.2 μl ROX qPCR Master Mix (2X, Fermentas, Germany) and 1 µl of forward and reverse primers (5 pM) adjusted to a total volume of 10 µl using nuclease-free water. The primer sequences, annealing temperatures and the size of amplified products are shown in table 1. Statistical analysis Statistical significance analysis was considered to be P